Translation initiation is a central biological process. It is also a key point in the regulation of gene expression. Eukaryotic translation initiation is a potential target of anticancer, antiviral and antifungal drugs. Attempts to develop drugs targeting translation initiation will be hampered, however, by the paucity of information about the molecular mechanics of this process. This proposal describes experiments aimed at understanding the molecular mechanisms underlying the recognition of the translation initiation site in an mRNA by the eukaryotic protein synthesis machinery. This is arguably the most important reading of the genetic code during gene expression because if it goes awry a miscoded protein will be produced. Upon recognition of the initiation codon, a signal is sent to the central G protein initiation factor elF2 to irreversibly hydrolyze its bound GTP and release the methionyl initiator tRNA into the P site of the smallribosomal subunit. This event is the first committed step in translation initiation; after it happens the complex must proceed with initiation at that point on the mRNA or abort the process. Thus the irreversible hydrolysis of GTP by elF2 must be regulated exquisitely carefully such that it does not happen at the wrong place on the mRNA but happens very rapidly at the right place. The molecular mechanics of the formation of the 43S[unreadable]mRNA pre-initiation complex, its identification of the start codon in the mRNA, and the triggering of irreversible GTP hydrolysis by elF2 will be elucidated by the experiments described in this proposal. The work will employ a reconstituted yeast-based translation initiation system. The roles and mechanisms of each of the key components of the initiation machinery required for these steps will be elucidated through a thermodynamic and kinetic dissection of the pathway. These studies will also use a number of mutant versions of initiation factors that produce well characterized phenotypes in vivo, which were isolated by our collaborators. These studies will thus synergistically harness the power of yeast genetics and molecular biology to the detailed biophysical and biochemical studies possible in vitro.